Description Embryonic stem cells (ESC) and induced pluripotent stem cells (IPSC) hold great promise for the treatment and study of debilitating diseases. Stem cells are also of considerable interest to cancer biologists because of evidence that many human cancers are caused by the aberrant expansion of cells with stem cell-like properties. Although considerable knowledge has been acquired, much remains to be learned about the fundamental molecular properties of stem cells and of the pluripotent state, which is defined as the capacity to differentiate into nearly all cell lineages. From a gene regulation perspective, most studies of pluripotency have focused on, 1. master transcriptional regulators of pluripotency and the gene networks controlled by these regulators, 2. bivalent histone modification domains that characterize the promoters of genes involved in early developmental decisions, and 3. fundamental differences in chromatin structure that distinguish pluripotent cells from differentiated cells. Recently, evidence has emerged from our lab and others that the marking of enhancers for typical tissue-specific genes by pioneer transcription factors and unmethylated CpG dinucleotides may also be critical for establishing or maintaining the pluripotency state. It has been hypothesized that these enhancer marks provide competence for transcriptional activation in differentiated cell types. The goals of the proposed research are to better understand how tissue-specific enhancers are marked in ESC and to rigorously examine the functional significance of these enhancer marks. In Aim 1, bacterial artificial chromosomes (BACs) will be used to identify and characterize DNA motifs and transcription factors that positively and negatively regulate the establishment of unmethylated windows observed in ESC and IPSC at well-characterized tissue-specific enhancers. By working closely with the other three project Pis, we will gain further insight into the relevance of the enhancer marks by characterizing their conservation between human and mouse ESC and IPSC, by examining the timing of their establishment during reprogramming, and by evaluating their organization within the overall 3-dimensional nuclear architecture of pluripotent cells. In Aim 2, the functional relevance of the enhancer marks will be examined through the conditional recruitment of repressive chromatin complexes capable of erasing the marks in ESC. In these experiments, we will test the hypothesis that erasure of the marks in ESC compromises transcriptional activation of the tissue-specific genes following differentiation. Finally, using the same tools that are generated for these experiments, we will assist Dr. Zaret with the goals of his project (Project 2) by evaluating the ability of pluripotency factors to gain access to silent chromatin assembled in vivo through the action of different repressive chromatin complexes. There will be no human or animal experimentation with the proposed work.